Heterocyclic compounds

ABSTRACT

Certain thienopyrrolyl and furanopyrrolyl compounds are disclosed as useful to treat or prevent disorders and conditions mediated by the histamine H 4  receptor, including allergic rhinitis.

FIELD OF THE INVENTION

[0001] The invention relates to novel, pharmaceutically active, fusedheterocyclic compounds and methods of using them to treat or preventdisorders and conditions mediated by the histamine H₄ receptor.

BACKGROUND OF THE INVENTION

[0002] Histamine was first identified as a hormone (G. Barger and H. H.Dale, J. Physiol. (London) 1910, 41:19-59) and has since beendemonstrated to play a major role in a variety of physiologicalprocesses, including the inflammatory “triple response” via H₁ receptors(A. S. F. Ash and H. O. Schild, Br. J. Pharmac. Chemother. 1966,27:427-439), gastric acid secretion via H₂ receptors (J. W. Black etal., Nature 1972, 236:385-390), and neurotransmitter release in thecentral nervous system via H₃ receptors (J. -M. Arrang et al., Nature1983, 302:832-837) (for review see S. J. Hill et al., Pharmacol. Rev.1997, 49(3):253-278). All three histamine receptor subtypes have beendemonstrated to be members of the superfamily of G protein-coupledreceptors (I. Gantz et al., Proc. Natl. Acad. Sci. U.S.A. 1991,88:429-433; T. W. Lovenberg et al., Mol. Pharmacol. 1999,55(6):1101-1107; M. Yamashita et al., Proc. Natl. Acad. Sci. U.S.A.1991, 88:11515-11519). There are, however, additional functions ofhistamine that have been reported, for which no receptor has beenidentified. For example, in 1994, Raible et al. demonstrated thathistamine and R-α-methylhistamine could activate calcium mobilization inhuman eosinophils (D. G. Raible et al., Am. J. Respir. Crit. Care Med.1994, 149:1506-1511). These responses were blocked by the H₃-receptorantagonist thioperamide. However, R-α-methylhistamine was significantlyless potent than histamine, which was not consistent with theinvolvement of known H₃ receptor subtypes. Therefore, Raible et al.hypothesized the existence of a novel histamine receptor on eosinophilsthat was non-H₁, non-H₂, and non-H₃. Most recently several groups (T.Oda et al., J. Biol. Chem. 2000, 275(47):36781-36786; C. Liu et al.,Mol. Pharmacol. 2001, 59(3):420-426; T. Nguyen et al., Mol. Pharmacol.2001, 59(3):427-433; Y. Zhu et al., Mol. Pharmacol. 2001, 59(3):434-441;K. L. Morse et al., J. Pharmacol. Exp. Ther. 2001, 296(3):1058-1066)have identified and characterized a fourth histamine receptor subtype,the H₄ receptor. This receptor is a 390 amino acid, seven-transmembrane,G protein-coupled receptor with approximately 40% homology to thehistamine H₃ receptor. In contrast to the H₃ receptor, which isprimarily located in the brain, the H₄ receptor is expressed at greaterlevels in neutrophils and mast cells, among other cells, as reported byMorse et al. (see above).

[0003] Events that elicit the inflammatory response include physicalstimulation (including trauma), chemical stimulation, infection, andinvasion by a foreign body. The inflammatory response is characterizedby pain, increased temperature, redness, swelling, reduced function, ora combination of these. Many conditions, such as allergies, asthma,chronic obstructed pulmonary disease (COPD), atherosclerosis, andautoimmune diseases, including rheumatoid arthritis and lupus, arecharacterized by excessive or prolonged inflammation. Inhibition ofleukocyte recruitment can provide significant therapeutic value.Inflammatory diseases or inflammation-mediated diseases or conditionsinclude, but are not limited to, acute inflammation, allergicinflammation, and chronic inflammation.

[0004] Mast cell de-granulation (exocytosis) leads to an inflammatoryresponse that may be initially characterized by a histamine-modulatedwheal and flare reaction. A wide variety of immunological (e.g.,allergens or antibodies) and non-immunological (e.g., chemical) stimulimay cause the activation, recruitment, and de-granulation of mast cells.Mast cell activation initiates allergic (H₁) inflammatory responses,which in turn cause the recruitment of other effector cells that furthercontribute to the inflammatory response. The histamine H2 receptorsmodulate gastric acid secretion, and the histamine H3 receptors affectneurotransmitter release in the central nervous system.

[0005] Examples of textbooks on the subject of inflammation include J.I. Gallin and R. Snyderman, Inflammation: Basic Principles and ClinicalCorrelates, 3^(rd) Edition, (Lippincott Williams & Wilkins,Philadelphia, 1999); V. Stvrtinova, J. Jakubovsky and I. Hulin,“Inflammation and Fever”, Pathophysiology Principles of Diseases(Textbook for Medical Students, Academic Press, 1995); Cecil et al.,Textbook Of Medicine, 18^(th) Edition (W. B. Saunders Company, 1988);and Steadmans Medical Dictionary.

SUMMARY OF THE INVENTION

[0006] The invention features a compound of formula (I):

[0007] Y is O or S;

[0008] Z is O or S;

[0009] n is 1 or 2;

[0010] m is 1 or 2;

[0011] n+m is2 or 3;

[0012] R¹ is H or C₁₋₆alkyl;

[0013] R² is H, F, Cl, Br or C₁₋₆alkyl;

[0014] R³ and R⁴ are, independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl,C₁₋₄alkyl(C₃₋₆cycloalkyl), cyano, —CF₃, —(CO)NR^(p)R^(q), —(CO)OR^(r),—CH₂NR^(p)R^(q) or —CH₂OR^(r); where R^(p), R^(q) and R^(r) areindependently selected from H, C₁₋₄alkyl, C₃₋₆cycloalkyl, phenyl,—C₁₋₂alkyl(C₃₋₆cycloalkyl), benzyl or phenethyl, or R^(p) and R^(q)taken together with the nitrogen to which they are attached, form a 4-7membered heterocyclic ring with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkylmoiety of the foregoing is optionally and independently substituted withbetween 1 and 3 substituents selected from C₁₋₃alkyl, halo, hydroxy,amino, and C₁₋₃alkoxy;

[0015] R⁵ and R⁶ are, independently, H or C₁₋₆alkyl;

[0016] R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—O—R^(a) or—R^(e)—N(R^(c))(R^(d)), where R^(a) is H, cyano, —(C═O)N(R^(c))(R^(d)),—C(═NH)(NH₂), C₁₋₁₀alkyl, C₂₋₈alkenyl, C₃₋₈cycloalkyl, C₄₋₇heterocyclicradical or phenyl, where the C₄₋₇heterocyclic radical is attached at acarbon atom and contains one of O, S, NH or NC₁₋₄alkyl, and optionallyan additional NH or NC₁₋₆alkyl in rings of 5 or 6 or 7 members, whereR^(b) is C₁₋₈alkylene or C₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene orC₂₋₈alkenylene, where R^(c) and R^(d) are each independently H,C₁₋₄alkyl, C₂₋₄alkenyl, C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d)taken together with the nitrogen to which they are attached, form a 4-7membered heterocyclic ring with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkylmoiety of the foregoing is optionally and independently substituted withbetween 1 and 3 substituents selected from C₁₋₃alkyl, halo, hydroxy,amino, and C₁₋₃alkoxy;

[0017] alternatively, R⁷ may be taken together with an adjacent R⁴ aswell as their carbon and nitrogen of attachment to form a 5, 6 or 7membered heterocyclic ring, with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and optionally and independentlysubstituted with between 1 and 3 substituents selected from C₁₋₃alkyl,halo, hydroxy, amino, and C₁₋₃alkoxy;

[0018] R⁸ and R⁹ are, independently, H, F, Cl, Br, I, C₁₋₄alkyl,C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl, —OCH₂Ph, —CF₃, —OCF₃,—SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl, —OH, phenyl, benzyl,phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k) (where R^(t) is H orC₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl, —(S═(O)_(p))—C₁₋₄alkyl (wherein p is0, 1 or 2), nitro, —SO₂NR^(l)R^(m) (wherein R^(l) and R^(m) areindependently selected from H, C₁₋₄alkyl, phenyl, benzyl or phenethyl,or R^(l) and R^(m) taken together with the nitrogen to which they areattached, form a 4-7 membered heterocyclic ring with 0 or 1 additionalheteroatoms selected from O, S, NH or NC₁₋₄alkyl), —(C═O)NR^(l)R^(m),cyano or phenyl, where any phenyl or alkyl or cycloalkyl moiety of theforegoing is optionally and independently substituted with between 1 and3 substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, andC₁₋₃alkoxy;

[0019] and enantiomers, diastereomers and pharmaceutically acceptablesalts and esters thereof,

[0020] with the following provisos,

[0021] that R⁶ adjacent to N must be H where R⁴ adjacent to N is otherthan H,

[0022] that R⁷ is not —CH₂CH₂OH; and

[0023] that where the core molecule is a 4H-furo, then one of R⁴ and R⁶adjacent to N must not be methyl when the other is hydrogen unless R⁶and R⁴ are taken together to form a bridging moiety.

[0024] The invention also features pharmaceutical compositionscontaining such compounds and methods of using such compositions in thetreatment or prevention of H₄-mediated diseases and conditions,particularly those wherein it is desirable to antagonize the H₄receptor.

DETAILED DESCRIPTION

[0025] Preferably, Y is S.

[0026] Preferably, Z is O.

[0027] Preferably, n is 1 and m is 1.

[0028] Preferrably, R¹ is selected from the group consisting of H ormethyl.

[0029] Preferrably, R² is H.

[0030] Preferrably, R³ and R⁴ are, independently, selected from thegroup consisting of

[0031] a) H,

[0032] b) —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, n-butyl, i-butyl,t-butyl,

[0033] c) cyclopropyl, cyclopentyl, cyclohexyl, —CH₂cyclopropyl,—CH₂cyclopentyl, —CH₂cyclohexyl, —CH₂Ocyclopropyl, —CH₂Ocyclopentyl,—CH₂Ocyclohexyl,

[0034] d) cyano,

[0035] e) trifluoromethyl,

[0036] f) —(C═O)NH₂, —(C═O)NHC₁₋₄alkyl, —(C═O)N(C₁₋₄alkyl)₂,—(C═O)NHphenyl, —(C═O)pyrrolidin-1-yl, —(C═O)imidazolidin-1-yl,—(C═O)pyrazolidin-1-yl, —(C═O)piperidin-1-yl, —(C═O)piperazin-1-yl,—(C═O)morpholin-4-yl, —(C═O)thiomorpholin-4-yl,

[0037] g) —COOH, —COOCH₃, —COOCH₂CH₃, —COOphenyl, —COObenzyl,

[0038] h) —CH₂NH₂, —CH₂NHC₁₋₄alkyl, —CH₂N(C₁₋₄alkyl)₂, —CH₂NHphenyl,—CH₂NHbenzyl, —CH₂pyrrolidin-1-yl, —CH₂imidazolidin-1-yl,—CH₂pyrazolidin-1-yl, —CH₂piperidin-1-yl, —CH₂piperazin-1-yl,—CH₂morpholin-4-yl, —CH₂thiomorpholin4-yl,

[0039] i) —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂OCH₃, —CH₂OCH₂CH₃,—CH₂OCH₂CH₂CH₃, —CH₂OCH(CH₃)₂, —CH₂O-n-butyl, —CH₂O-i-butyl,—CH₂O-t-butyl, —CH₂Ophenyl, —CH₂Obenzyl and —CH₂OCH₂cyclopropyl.

[0040] Most preferrably, R³ and R⁴ are, independently, H or —CH₃.

[0041] Preferrably, R⁵ and R⁶ are, independently, selected from thegroup consisting of H and methyl.

[0042] Most preferrably, R⁵ and R⁶ are H.

[0043] Preferrably, R⁷ is selected from the group consisting of

[0044] a) H, —CH₂CH₂CH₂OH,

[0045] b) cyano,

[0046] c) —(C═O)NH₂, —(C═O)NHC₁₋₄alkyl, —(C═O)N(C₁₋₄alkyl)₂,—(C═O)NHphenyl, —(C═O)pyrrolidin-1-yl, —(C═O)imidazolidin-1-yl,—(C═O)pyrazolidin-1-yl, —(C═O)piperidin-1-yl, —(C═O)piperazin-1-yl,—(C═O)morpholin-4-yl, —(C═O)thiomorpholin-4-yl, —CH₂(C═O)NH₂,—CH₂(C═O)NHC₁₋₄alkyl, —CH₂(C═O)N(C₁₋₄alkyl)₂, —CH₂(C═O)NHphenyl,—CH₂(C═O)pyrrolidin-1-yl, —CH₂(C═O)imidazolidin-1-yl,—CH₂(C═O)pyrazolidin-1-yl, —CH₂(C═O)piperidin-1-yl,—CH₂(C═O)piperazin-1-yl, —CH₂(C═O)morpholin-4-yl,—CH₂(C═O)thiomorpholin-4-yl, —CH₂CH₂O(C═O)NH₂, —CH₂CH₂O(C═O)NHC₁₋₄alkyl,—CH₂CH₂O(C═O)N(C₁₋₄alkyl)₂, —CH₂CH₂O(C═O)NHphenyl,—CH₂CH₂O(C═O)pyrrolidin-1-yl, —CH₂CH₂O(C═O)imidazolidin-1-yl,—CH₂CH₂O(C═O)pyrazolidin-1-yl, —CH₂CH₂O(C═O)piperidin-1-yl,—CH₂CH₂O(C═O)piperazin-1-yl, —CH₂CH₂O(C═O)morpholin-4-yl,—CH₂CH₂O(C═O)thiomorpholin-4-yl,

[0047] d) —C(═NH)(NH₂), —CH₂C(═NH)(NH₂),

[0048] e) —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, n-butyl, i-butyl,t-butyl, —CH₂CH₂OCH₃, —CH₂CH₂OCH₂CH₃, —CH₂CH₂OCH₂CH₂CH₃,—CH₂CH₂OCH(CH₃)₂, —CH₂CH₂O-n-butyl, —CH₂CH₂O-i-butyl, —CH₂CH₂O-t-butyl,

[0049] f), —CH₂CH═CH₂,

[0050] g) cyclopropyl, cyclopentyl, cyclohexyl, —CH₂cyclopropyl,—CH₂cyclopentyl, —CH₂cyclohexyl, —CH₂CH₂Ocyclopropyl,—CH₂CH₂Ocyclopentyl, —CH₂CH₂Ocyclohexyl,

[0051] h) pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl,piperazinyl, morpholinyl, thiomorpholinyl, —CH₂pyrrolidinyl,—CH₂imidazolidinyl, —CH₂pyrazolidinyl, —CH₂piperidinyl, —CH₂piperazinyl,—CH₂morpholinyl, —CH₂thiomorpholinyl,

[0052] i) —CH₂CH₂NH₂, —CH₂CH₂NHC₁₋₄alkyl, —CH₂CH₂N(C₁₋₄alkyl)₂,—CH₂CH₂NHphenyl, —CH₂CH₂pyrrolidin-1-yl, —CH₂CH₂imidazolidin-1-yl,—CH₂CH₂pyrazolidin-1-yl, —CH₂CH₂piperidin-1-yl, —CH₂CH₂piperazin-1-yl,—CH₂CH₂morpholin4-yl, —CH₂CH₂thiomorpholin-4-yl,

[0053] j) phenyl, benzyl, phenethyl and benzyloxymethyl.

[0054] Most preferrably, R⁷ is selected from the group consisting of Hand —CH₃.

[0055] Preferred R⁷ taken together with an adjacent R⁴ as well as theircarbon and nitrogen of attachment are pyrrolidin-1,2-yl,imidazolidin-1,2-yl, imidazolidin-1,5-yl, pyrazolidin-1,5-yl,piperidin-1,2-yl, piperazin-1,2-yl, morpholin-4,5-yl andthiomorpholin-4,5-yl.

[0056] Most preferred R⁷ taken together with an adjacent R⁴ as well astheir carbon and nitrogen of attachment are pyrrolidin-1,2-yl andpiperidin-1,2-yl.

[0057] Preferrably, R⁸ and R⁹ are, independently, selected from thegroup consisting of H, —F, —Cl, —Br, —I, —CH₃, —CH₂CH₃, —OCH₃, —OCH₂CH₃,—OCH(CH₃)₂, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,—Ocyclopentyl, —Ocyclohexyl, —CF₃, —OCF₃, —SCF₃, —COOH, —COOCH₃,—COOCH₂CH₃, —C(O)CH₃, —NHCOCH₃, —NCH₃COCH₃, —NHSO₂CH₃, —NCH₃SO₂CH₃,—SOCH₃, —SO₂CH₃, —NO₂, —SO₂NH₂, —SO₂NHCH₃, —SO₂N(CH₃)₂, —C(O)NH₂,—C(O)N(CH₃)₂, —C(O)NH(CH₃), —CN and phenyl.

[0058] Most preferrably, R⁸ and R⁹ are, independently, selected from thegroup consisting of hydrogen, methyl, chloro and bromo. Further, it ismost preferred that one or both of R⁸ and R⁹ are not hydrogen.

[0059] The “pharmaceutically acceptable salts and esters thereof” referto those salt and ester forms of the compounds of the present inventionwhich that would be apparent to the pharmaceutical chemist, i.e., thosewhich that are non-toxic and which that would favorably affect thepharmacokinetic properties of said compounds of the present invention.Those compounds having favorable pharmacokinetic properties would beapparent to the pharmaceutical chemist, i.e., those which that arenon-toxic and which that possess such pharmacokinetic properties toprovide sufficient palatability, absorption, distribution, metabolismand excretion. Other factors, more practical in nature, which that arealso important in the selection, are cost of raw materials, ease ofcrystallization, yield, stability, hygroscopicity, and flowability ofthe resulting bulk drug. In addition, acceptable salts of carboxylatesinclude sodium, potassium, calcium and magnesium. Examples of suitablecationic salts include hydrobromic, hydroiodic, hydrochloric,perchloric, sulfuric, maleic, fumaric, malic, tartatic, citric, benzoic,mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic,pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamicand saccharic. Examples of suitable esters include such esters where oneor more carboxyl substituents is replaced withp-methoxybenzyloxycarbonyl, 2,4,6-trimethylbenzyloxycarbonyl,9-anthryloxycarbonyl, CH₃SCH₂COO—, tetrahydrofur-2-yloxycarbonyl,tetrahydropyran-2-yloxycarbonyl, fur-2-uloxycarbonyl,benzoylmethoxycarbonyl, p-nitrobenzyloxycarbonyl,4-pyridylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl,2,2,2-tribromoethoxycarbonyl, t-butyloxycarbonyl, t-amyloxycarbonyl,diphenylmethoxycarbonyl, triphenylmethoxycarbonyl, adamantyloxycarbonyl,2-benzyloxyphenyloxycarbonyl, 4-methylthiophenyloxycarbonyl, ortetrahydropyran-2-yloxycarbonyl.

[0060] The provisos are based on a failure to find activity in at leastone compound meeting the specifications of each proviso.

[0061] Preferred compounds of Formula I are compounds selected from thegroup consisting of:

[0062] Additional preferred compounds of Formula I are compoundsselected from the group consisting of:

[0063] Additional preferred compounds of Formula I are compoundsselected from the group consisting of:

[0064] Still further preferred compounds are made according to thesynthetic methods outlined in Schemes 1-4 where Y is S and selected fromthe group consisting of:

[0065] EX Compound

[0066] 26(2,3-Dimethyl-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0067] 27(2-Chloro-3-methyl-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0068] 28(3-Chloro-2-methyl-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0069] 29(2-Bromo-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0070] 30(3-Bromo-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0071] 31(4-Methyl-piperazin-1-yl)-(2-phenyl-6H-thieno[2,3-b]pyrrol-5-yl)-methanone;

[0072] 32[2-(4-Chloro-phenyl)-6H-thieno[2,3-b]pyrrol-5-yl]-(4-methyl-piperazin-1-yl)-methanone;

[0073] 33(3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(3,4-dimethyl-piperazin-1-yl)-methanone;

[0074] 34(3,4-Dimethyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone;

[0075] 35(2-Bromo-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0076] 36(3-Bromo-2-chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0077] 37(2,3-Dichloro-4H-thieno[3,2-b]pyrrol-5-yl)-(3-methyl-piperazin-1-yl)-methanone;

[0078] 38(4-Methyl-piperazin-1-yl)-(2-phenyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone;and

[0079] 39(4-Methyl-piperazin-1-yl)-[2-(4-trifluoromethyl-phenyl)-4H-thieno[3,2-b]pyrrol-5-yl]-methanone.

[0080] The following terms are defined below, and by their usagethroughout the disclosure.

[0081] “Alkyl” includes straight chain and branched hydrocarbons with atleast one hydrogen removed to form a radical group. Alkyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,1-methylpropyl, pentyl, isopentyl, sec-pentyl, hexyl, heptyl, octyl, andso on. Alkyl does not include cycloalkyl.

[0082] “Alkenyl” includes straight chain and branched hydrocarbonradicals as e with at least one carbon-carbon double bond (sp²).Alkenyls include ethenyl (or vinyl), prop-1-enyl, prop-2-enyl (orallyl), isopropenyl (or 1-methylvinyl), but-1-enyl, but-2-enyl,butadienyls, pentenyls, hexa-2,4-dienyl, and so on. Alkenyl does notinclude cycloalkenyl.

[0083] “Alkoxy” includes a straight chain or branched alkyl group with aterminal oxygen linking the alkyl group to the rest of the molecule.Alkoxy includes methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy,pentoxy and so on. “Aminoalkyl”, “thioalkyl”, and “sulfonylalkyl” areanalogous to alkoxy, replacing the terminal oxygen atom of alkoxy with,respectively, NH (or NR), S, and SO₂.

[0084] “Cycloalkyl” includes cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, and so on.

[0085] “Halo” includes fluoro, chloro, bromo, and iodo, and preferablyfluoro or chloro.

[0086] “Patient” or “subject” includes mammals such as humans andanimals (dogs, cats, horses, rats, rabbits, mice, non-human primates) inneed of observation, experiment, treatment or prevention in connectionwith the relevant disease or condition. Preferably, the patient is ahuman.

[0087] “Composition” includes a product comprising the specifiedingredients in the specified amounts as well as any product that resultsdirectly or indirectly from combinations of the specified ingredients inthe specified amounts.

[0088] The compounds as described above may be made according toprocesses within the skill of the art and/or that are described in theschemes and examples that follow. To obtain the various compoundsherein, starting materials may be employed that carry the ultimatelydesired substituents though the reaction scheme with or withoutprotection as appropriate. Alternatively, it may be necessary to employ,in the place of the ultimately desired substituent, a suitable groupthat may be carried through the reaction scheme and replaced asappropriate with the desired substituent.

[0089] Referring to Scheme 1, there are disclosed the following notesand additions. Various R¹ may be obtained from E1 or C1 by treatmentwith a base and and an appropriate alkylating agent. Where R² is halo,it may be obtained by treatment of E1 and C1 with an appropriatehalogenating agent. Where R² is alkyl, it may be obtained by replacingthe aldehyde of A1 with a ketone. P may be an alkyl, aryl or benzyl.Suitable bases include NaOEt, LDA, NaH, DBU, etc. The conversion of B1to C1 is thermolytic with typical temperatures ranging from 80 to 200°C. Suitable solvents for the conversion of B1 to C1 are xylene, cumene,diphenylether, etc. Acidic or basic hydrolysis will providedeprotection. In the case where P is benzyl, hydrogenolysis is alsouseful for deprotection. Typical coupling reagents for the conversion ofD1 to E1 include EDCl, HBTU, etc. Typical chlorination agents for theconversion of D1 to E1 include oxalyl chloride and thionyl chloride. Xis a halogenating agent such as Cl₂, N-bromosuccinimide, TAS-F, Br₂,N-chlorosuccinimide, etc.

[0090] Referring to Scheme 2, there are disclosed the following notesand additions. Various R¹ may be obtained from E2 or C2 by treatmentwith a base and and an appropriate alkylating agent. Where R² is halo,it may be obtained by treatment of E2 and C2 with an appropriatehalogenating agent. Where R² is alkyl, it may be obtained by replacingthe aldehyde of A2 with a ketone. P may be an alkyl, aryl or benzyl.Suitable bases include NaOEt, LDA, NaH, DBU, etc. The conversion of B2to C2 is thermolytic with typical temperatures ranging from 80 to 200°C. Suitable solvents for the conversion of B2 to C2 are xylene, cumene,diphenylether, etc. Acidic or basic hydrolysis will providedeprotection. In the case where P is benzyl, hydrogenolysis is alsouseful for deprotection. Typical coupling reagents for the conversion ofD2 to E2 include EDCl, HBTU, etc. Typical chlorination agents for theconversion of D2 to E2 include oxalyl chloride and thionyl chloride. Xis a halogenating agent such as Cl₂, N-bromosuccinimide, TAS-F, Br₂,N-chlorosuccinimide, etc.

[0091] Referring to Scheme 3, there are disclosed the following notesand additions. X is a halogenating agent such as Cl₂,N-bromosuccinimide, TAS-F, Br₂, N-chlorosuccinimide, etc.

[0092] Referring to Scheme 4, there are disclosed the following notesand additions. Typical bases include n-BuLi, LDA, t-BuLi, KHMDS.

[0093] The expression of the H₄ receptor in immune cells, including someleukocytes and mast cells, establishes it as an important target fortherapeutic intervention in a range of immunological and inflammatorydisorders (such as allergic, chronic, or acute inflammation).Specifically H₄ receptor ligands are expected to be useful for thetreatment or prevention of various mammalian disease states.

[0094] Thus, according to the invention, the disclosed compounds, whereantagonists of the H₄ receptor, and compositions are useful for theamelioration of symptoms associated with, the treatment of, and theprevention of, the following conditions and diseases: inflammatorydisorders, asthma, psoriasis, rheumatoid arthritis, ulcerative colitis,Crohn's disease, inflammatory bowel disease, multiple sclerosis,allergic disorders, allergic rhinitis, dermatological disorders,autoimmune disease, lymphatic disorders, atherosclerosis, andimmunodeficiency disorders. The disclosed compounds may also be usefulas adjuvants in chemotherapy or in the treatment of itchy skin.

[0095] Aspects of the invention include (a) a pharmaceutical compositioncomprising a compound of formula (I), or one or more preferred compoundsas described herein, and a pharmaceutically acceptable carrier; (b) apackaged drug comprising (1) a pharmaceutical composition comprising acompound of formula (I) and a pharmaceutically acceptable carrier, and(2) instructions for the administration of said composition for thetreatment or prevention of an H₄-mediated disease or condition.

[0096] The invention also provides a method for treating an H₄-mediatedcondition in a patient, said method comprising administering to thepatient a pharmaceutically effective amount of a composition comprisinga compound of formula (I) and other disclosed or preferred compounds.For example, the invention features a method for treating an H₄ mediatedcondition in a patient, said method comprising administering to thepatient a pharmaceutically effective H₄-antagonizing amount of acomposition comprising a compound of formula (I).

[0097] The effect of an antagonist may also be produced by an inverseagonist. Inverse agonism describes the property of a compound toactively turn off a receptor that displays constitutive activity.Constitutive activity can be identified in cells that have been forcedto over-express the human H₄ receptor. Constitutive activity can bemeasured by examining cAMP levels or by measuring a reporter genesensitive to cAMP levels after a treatment with a cAMP-stimulating agentsuch as forskolin. Cells that over-express H₄ receptors will displaylower cAMP levels after forskolin treatment than non-expressing cells.Compounds that behave as H₄ agonists will dose-dependently lowerforskolin-stimulated cAMP levels in H₄-expressing cells. Compounds thatbehave as inverse H₄ agonists will dose-dependently stimulate cAMPlevels in H₄-expressing cells. Compounds that behave as H₄ antagonistswill block either H₄ agonist-induced inhibition of cAMP or inverse H₄agonist-induced increases in cAMP.

[0098] Further embodiments of the invention include disclosed compoundsthat are inhibitors of a mammalian histamine H₄ receptor function,inhibitors of inflammation or inflammatory responses in vivo or invitro, modulators of the expression of a mammalian histamine H₄ receptorprotein, inhibitors of polymorphonuclear leukocyte activation in vivo orin vitro, or combinations of the above, and corresponding methods oftreatment, prophylaxis, and diagnosis comprising the use of a disclosedcompound.

[0099] Those skilled in the art will be able to determine, according toknown methods, the appropriate dosage for a patient, taking into accountfactors such as age, weight, general health, the type of symptomsrequiring treatment, and the presence of other medications. In general,an effective amount will be between 0.01 and 1000 mg/kg per day,preferably between 0.5 and 300 mg/kg body weight, and daily dosages willbe between 10 and 5000 mg for an adult subject of normal weight.Capsules, tablets or other formulations (such as liquids and film-coatedtablets) may be of between 0.5 and 200 mg, such as 1, 3, 5, 10, 15, 25,35, 50 mg, 60 mg, and 100 mg and can be administered according to thedisclosed methods.

[0100] Dosage unit forms include tablets, capsules, pills, powders,granules, aqueous and nonaqueous oral solutions and suspensions, andparenteral solutions packaged in containers adapted for subdivision intoindividual doses. Dosage unit forms can also be adapted for variousmethods of administration, including controlled release formulations,such as subcutaneous implants. Administration methods include oral,rectal, parenteral (intravenous, intramuscular, subcutaneous),intracisternal, intravaginal, intraperitoneal, intravesical, local(drops, powders, ointments, gels or cream), and by inhalation (a buccalor nasal spray).

[0101] Parenteral formulations include pharmaceutically acceptableaqueous or nonaqueous solutions, dispersion, suspensions, emulsions, andsterile powders for the preparation thereof. Examples of carriersinclude water, ethanol, polyols (propylene glycol, polyethylene glycol),vegetable oils, and injectable organic esters such as ethyl oleate.Fluidity can be maintained by the use of a coating such as lecithin, asurfactant, or maintaining appropriate particle size. Carriers for soliddosage forms include (a) fillers or extenders, (b) binders, (c)humectants, (d) disintegrating agents, (e) solution retarders, (f)absorption accelerators, (g) adsorbants, (h) lubricants, (i) bufferingagents, and (j) propellants.

[0102] Compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents; antimicrobial agents suchas parabens, chlorobutanol, phenol, and sorbic acid; isotonic agentssuch as a sugar or sodium chloride; absorption-prolonging agents such asaluminum monostearate and gelatin; and absorption-enhancing agents.

EXAMPLES General Synthetic Procedures

[0103] Procedure A: Annulation of Aldehyde with Ethyl Azidoacetate

[0104] A solution of aldehyde A1, A2 or A3 (1 equiv) and ethylazidoacetate (4 equiv) was added dropwise to a solution of NaOEt (4equiv) in EtOH (0.15 M) at 0° C. The reaction mixture was stirred at 0°C. for 1 h and at room temperature for an additional 1 h. The reactionmixture was then poured into satd aq NH₄Cl and extracted with ether. Thecombined organics were dried (Na₂SO₄) and concentrated in vacuo. Theresidue was purified by silica gel column chromatography to provide thedesired acrylate.

[0105] A solution of the resultant acrylate in xylene (0.2 M) was heatedat 145° C. for 10-60 min and then allowed to cool to room temperature.The xylene solution was either cooled further to induce productcrystallization or directly subjected to silica gel columnchromatography to obtain the desired annulation product.

[0106] Procedure B: Ester Hydrolysis

[0107] A solution (0.2 M) of the ethyl ester (1 equiv, from Procedure A)and LiOH (5 equiv) in THF/MeOH/H₂O (3:1:1) was heated at 65° C.overnight, cooled to room temperature, acidified with 2 N HCl, andextracted with EtOAc. The organic layer was separated, dried overNa₂SO₄, and concentrated to give the desired crude acid, which was takento the next step without further purification.

[0108] Procedure C: Amide Formation Using1-(3-Dimethylaminopropyl)-3-ethylcarbodimide Hydrochloride (EDCl)

[0109] A mixture of acid (1 equiv, from Procedure B), amine (1.5 equiv)and EDCl (2.0 equiv) in CH₂Cl₂ (0.2 M) was stirred at room temperatureovernight and then partitioned between CH₂Cl₂ and satd aq NaHCO₃. Theorganic layer was separated, washed with H₂O, dried over Na₂SO₄, andconcentrated. The crude product was further purified by silica gelcolumn chromatography.

[0110] Procedure D: Amide Formation via Acyl Chloride Intermediate

[0111] A mixture of acid (1 equiv, from Procedure B) in CH₂Cl₂ (0.5 M)was treated at 0° C. with oxalyl chloride (1.2 equiv) followed by 1-2drops of DMF. The reaction mixture was stirred at 0° C. for 30 min thenslowly warmed to room temperature and stirred for an additional 1 h. Allvolatiles were removed to provide the crude acyl chloride.

[0112] The resultant acyl chloride was treated with amine (5.0 equiv) inCH₂Cl₂ (0.2 M) and allowed to stir at room temperature for 3 h. Thereaction mixture was partitioned between CH₂Cl₂ and satd aq NaHCO₃. Theorganic layer was separated, washed with H₂O, dried over Na₂SO₄, andconcentrated. The crude product was further purified with silica gelcolumn chromatography.

General Analytical Procedures

[0113] NMR spectra were obtained on either a Bruker model DPX400 (400MHz) or DPX500 (500 MHz) spectrometer. The format of the 1H NMR databelow is: chemical shift in ppm down field of the tetramethylsilanereference (multiplicity, coupling constant J in Hz, integration).

[0114] Mass spectra were obtained on a Hewlett Packard (Agilent) series1100 MSD using electrospray ionization (ESI) in either positive ornegative mode as indicated. The “mass calculated” for a molecularformula is the monoisotopic mass of the compound.

[0115] Silica Gel Column Chromatography:

[0116] Normal-phase column chromatography was accomplished using an ISCOFoxy 200 system employing one of the following commercially availableprepacked columns: ISCO Redisep (SiO₂, 10 g, 12 g, 35 g, 40 g, or 120g).

Example 1

[0117]

(4-Methyl-piperazin-1-yl)-(6H-thieno[2,3-b]pyrrol-5-yl)-methanone

[0118] A. 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester.Thiophene-3-carbaldehyde (2.24 g, 20 mmol) was annulated according toProcedure A to provide the title compound (1.2 g, 31%) as a white solid.TLC (silica, 20% EtOAc/hexanes): R_(f)=0.50. ¹H NMR (CDCl₃, 400 MHz):10.30 (brs, 1H), 7.10 (d, J=1.9 Hz, 1H), 6.96 (d, J=5.4 Hz, 1H), 6.87(d, J=5.4 Hz, 1H), 4.39 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H).

[0119] B. 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid.6H-Thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (835 mg, 4.3 mmol)was hydrolyzed according to Procedure B to provide the crude acid as apale-yellow solid. ¹H NMR (CD₃OD, 400 MHz): 7.02 (s, 1H), 6.96 (s, 1H),6.95 (s, 1H).

[0120] C.(4-Methyl-piperazin-1-yl)-(6H-thieno[2,3-b]pyrrol-5-yl)-methanone.6H-Thieno[2,3-b]pyrrole-5-carboxylic acid (60 mg, 0.35 mmol) was coupledwith N-methylpiperazine according to Procedure C to provide the titlecompound (44 mg, 50%) as a light yellow solid. TLC (silica, 10%MeOH/CH₂Cl₂): R_(f)=0.4. MS (electrospray): exact mass calculated forC₁₂H₁₅N₃OS, 249.09; m/z found, 250.1 [M+H]⁺. ¹H NMR (CD₃OD, 400 MHz, TFAsalt): 6.97 (s, 1H), 6.96 (s, 1H), 6.85 (s, 1H), 4.20−3.10 (m, 8H), 2.96(s, 3H).

Example 2

[0121]

(Hexahydro-pyrrolo[1,2-a]pyrazin-2-yl)-(6H-thieno[2,3-b]pyrrol-5-yl)-methanone

[0122] 6H-Thieno[2,3-b]pyrrole-5-carboxylic acid (60 mg, 0.35 mmol) wascoupled with octahydro-pyrrolo[1,2-a]pyrazine according to Procedure Cto provide the title compound (34 mg, 35%) as a light yellow solid. TLC(silica, 10% MeOH/CH₂Cl₂): R_(f)=0.4. MS (electrospray): exact masscalculated for C₁₄H₁₇N₃OS, 275.11; m/z found, 276.2 [M+H]⁺. ¹H NMR(CDCl₃, 400 MHz): 11.1 (br s, 1H), 6.96 (d, J=5.4 Hz, 1H), 6.87 (d,J=5.4 Hz, 1H), 6.71 (d, J=1.9 Hz, 1H), 4.84 (d, J=12.2 Hz, 1H), 4.70 (d,J=12.2 Hz, 1H), 3.30−2.90 (m, 4H), 2.30−1.40 (m, 7H).

Example 3

[0123]

(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0124] A. 2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethylester. A solution of 6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethylester (580 mg, 3.0 mmol) in acetic acid (6 mL) and CHCl₃ (6 mL) wastreated with three portions of N-chlorosuccinimide (total 415 mg, 3.15mmol) at 0° C. over 2 h. The reaction mixture was slowly warmed to roomtemperature and stirred overnight. The CHCl₃ was then removed, and theresidue was basified with 4 N NaOH and extracted with EtOAc. Thecombined organics were washed with satd aq NaHCO₃, dried over Na₂SO₄,and concentrated. Column chromatography (SiO₂, 5-10% EtOAc/hexanes) gave600 mg (88%) of a white solid. TLC (silica, 20% EtOAc/hexanes):R_(f)=0.5. ¹H NMR (CDCl₃, 400 MHz): 10.5 (br s, 1H), 6.97 (d, J=2.0 Hz,1H), 6.85 (s, 1H), 4.39 (q, J=7.2 Hz, 2H), 1.35 (t, J=7.2 Hz, 3H).

[0125] B.(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester (102 mg,0.45 mmol) was hydrolyzed (Procedure B) and coupled withN-methylpiperazine (procedure D) to provide the title compound (102 mg,80% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₂H₁₄ClN₃OS,283.05; m/z found, 284.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.5 (br s,1H), 6.87 (s, 1H), 6.61 (d, J=1.8 Hz, 1H), 3.92 (t, J=5.1 Hz, 4H), 2.50(t, J=5.1 Hz, 4H), 2.35 (s, 3H).

Example 4

[0126]

(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(hexahydro-pyrrolo[1,2-a]pyrazin-2-yl)-methanone

[0127] 2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester(102 mg, 0.45 mmol) was hydrolyzed (Procedure B) and then coupled withoctahydro-pyrrolo[1,2-a]pyrazine (Procedure D) to provide the titlecompound (108 mg, 78% for two steps) as an off-white solid. TLC (silica,10% MeOH/CH₂Cl₂): R_(f)=0.35. MS (electrospray): exact mass calculatedfor C₁₄H₁₆ClN₃OS, 309.07; m/z found, 310.1 [M+H]⁺. ¹H NMR (CDCl₃, 400MHz): 11.1 (br s, 1H), 6.86 (s, 1H), 6.62 (s, 1H), 4.79 (d, J=11.8 Hz,1H), 4.67 (d, J=11.8 Hz, 1H), 3.30−2.90 (m, 4H), 2.30−1.40 (m, 7H).

Example 5

[0128]

(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-piperazin-1-yl-methanone

[0129] 2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester(102 mg, 0.45 mmol) was hydrolyzed (Procedure B) and then coupled withpiperazine (Procedure D) to provide the title compound (42 mg, 35% fortwo steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.15. MS (electrospray): exact mass calculated for C₁₁H₁₂ClN₃OS,269.04; m/z found, 270.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.5 (brs,1H), 6.87 (s, 1H), 6.61 (s,1H), 3.87 (t, J=4.8 Hz, 4H), 2.96 (t, J=5.2Hz, 4H).

Example 6

[0130]

(4H-Furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0131] A. 4H-Furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester.Furan-2-carbaldehyde (1.92 g, 20 mmol) was annulated according toprocedure A to provide the title compound (1.97 g, 55%) as a whitesolid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.50. ¹H NMR (CDCl₃, 400MHz): 8.95 (br s,1H), 7.51 (d, J=2.2 Hz, 1H), 6.81−6.80 (m, 1H),6.46−6.45 (m, 1H), 4.35 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H).

[0132] B.(4H-Furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.4H-Furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg, 1.12 mmol)was hydrolyzed (Procedure B) and coupled with N-methylpiperazine(Procedure D) to provide the title compound (185 mg, 71% for two steps)as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂): R_(f)=0.4. MS(electrospray): exact mass calculated for C₁₂H₁₅N₃O₂, 233.12; m/z found,234.2 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.3 (br s, 1H), 7.43 (d, J=2.2Hz, 1H), 6.43−6.42 (m, 2H), 3.90 (t, J=5.0 Hz, 4H), 2.47 (t, J=5.1 Hz,4H), 2.32 (s, 3H).

Example 7

[0133]

(4-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone

[0134] A. 4H-Thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester. To asolution of thiophene-2-carbaldehyde (1.10 mL, 11.7 mmol) and ethylazidoacetate (1.4 mL, 11.7 mmol) in EtOH (35 mL) cooled to 0° C. wasadded NaOEt (1.0 g, 14.7 mmol) in one portion. The mixture was allowedto reach room temperature over 14 h and was then poured into water (400mL) and extracted with CH₂Cl₂ (3×50 mL). The combined organics werewashed with water and brine, dried over Na₂SO₄, and concentrated. Theresidue was taken up in xylenes (10 mL), and the resulting solution wasrefluxed for 1 h. The solution was cooled and then loaded directly ontosilica gel and purified (35 g SiO₂, 10-20% EtOAc/hexanes) to reveal 0.12g (5%) of a yellowish solid. ¹H NMR (400 MHz, CDCl₃): 9.06 (br s, 1H),7.33 (d, J=5.3 Hz, 1H), 7.15−7.14 (m, 1H), 6.96 (dd, J=5.3, 0.8 Hz, 1H),4.37 (q, J=7.3 Hz, 2H), 1.39 (t, J=7.3Hz, 3H). ¹³C NMR (100 MHz, CDCl₃):161.3, 140.9, 129.2, 126.9, 124.6, 110.9, 107.3, 60.4, 14.2.

[0135] B.(4-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone. To asolution of 4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (98.5mg, 0.50 mmol) in wet THF (3 mL) was added LiOH (129 mg, 3 mmol). Thismixture was stirred at room temperature for 3 days. The reaction mixturewas diluted with water (50 mL), and 1 M HCL was added to adjust the pHto about 3. This mixture was then extracted with EtOAc, and the combinedorganics were dried over Na₂SO₄. The solvent was removed to reveal 73.4mg (87%) of the free acid, which was used in the coupling event withoutfurther purification. The acid (73.4 mg, 0.44 mmol) was taken up in THF(3 mL), and CDI (87.1 mg, 0.54 mmol) was added in one portion. Thereaction mixture was stirred for 1 h. To this mixture was then added1-methylpiperazine (70 μL), and the mixture stirred for an additional 6h. The reaction mixture was diluted with EtOAc, washed with water,NaHCO₃ (aq) and then brine, and subsequently purified by columnchromatography (10 g SiO₂, 1-8% MeOH (2 M NH₃)/CH₂Cl₂) to reveal 55.6 mg(51%) of the title compound. ¹H NMR (400 MHz, CDCl₃): 9.26 (br s, 1H),7.26 (d, J=5.3 Hz, 1H), 6.97 (dd, J=5.3, 0.8 Hz, 1H), 6.75−6.74 (m, 1H),4.07−3.88 (m, 4H), 2.68−2.48 (m, 4H), 2.43 (br s, 3H). MS(electrospray): exact mass calculated for C₁₂H₁₅N₃OS, 249.09; m/z found,250.1 [M+H]⁺.

Example 8

[0136]

Piperazin-1-yl-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone

[0137] 4H-Thieno[3,2-b]pyrrole-5-carboxylic acid (50 mg, 0.30 mmol) wascoupled with piperazine according to Procedure D to provide the titlecompound (25 mg, 35%) as an off-white solid. TLC (silica, 10%MeOH/CH₂Cl₂): R_(f)=0.15. MS (electrospray): exact mass calculated forC₁₁H₁₃N₃OS, 235.08; m/z found, 236.1 [M+H]⁺. ¹H NMR (CD₃OD, 400 MHz):7.33 (d, J=5.3 Hz, 1H), 6.98 (dd, J=5.2, 0.7 Hz, 1H), 6.89 (d, J=0.6 Hz,1H), 4.08 (t, J=5.3 Hz, 4H), 3.50−3.20 (m, 4H).

Example 9

[0138]

(3-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-yl)-methanone

[0139] 4H-Thieno[3,2-b]pyrrole-5-carboxylic acid (50 mg, 0.30 mmol) wascoupled with 2-methylpiperazine according to Procedure D to provide thetitle compound (58 mg, 78%) as an off-white solid. TLC (silica, 10%MeOH/CH₂Cl₂): R_(f)=0.15. MS (electrospray): exact mass calculated forC₁₂H₁₅N₃OS, 249.09; m/z found, 250.1 [M+H]⁺. ¹H NMR (CD₃OD, 400 MHz):7.33 (d, J=5.3 Hz, 1H), 6.98 (d, J=5.3 Hz, 1H), 6.88 (s, 1H), 4.62−4.56(m, 2H), 3.50−3.20 (m, 5H), 1.36 (d, J=6.6 Hz, 3H).

Example 10

[0140]

(2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0141] A. 2-Chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethylester. 5-Chloro-thiophene-2-carbaldehyde (2.92 g, 20 mmol) was annulatedaccording to Procedure A to provide the title compound (2.8 g, 61%) as awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.48. ¹H NMR (CDCl₃,400 MHz): 9.10 (br s, 1H), 7.04 (dd, J=1.9, 0.7 Hz, 1H), 6.89 (d, J=0.7Hz, 1H), 4.37 (q, J=7.2 Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

[0142] B.(2-Chloro-4H-thieno[3.2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2-Chloro-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (230 mg,1.0 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure C) to provide the title compound (128 mg,45% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₂H₁₄ClN₃OS,283.05; m/z found, 284.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.1 (br s,1H), 6.88 (s, 1H), 6.64 (d, J=1.4 Hz, 1H), 3.91 (t, J=4.4 Hz, 4H), 2.49(t, J=5.1 Hz, 4H), 2.35 (s, 3H).

Example 11

[0143]

(2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(hexahydro-pyrrolo[1,2-a]pyrazin-2-yl)-methanone

[0144] 2-Chloro4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester(230 mg, 1.0 mmol) was hydrolyzed (Procedure B) and then coupled withoctahydro-pyrrolo[1,2-a]pyrazine (Procedure C) to provide the titlecompound (93 mg, 30% for two steps) as an off-white solid. TLC (silica,10% MeOH/CH₂Cl₂): R_(f)=0.4. MS (electrospray): exact mass calculatedfor C₁₄H₁₆ClN₃OS, 309.07; m/z found, 310.1 [M+H]⁺. ¹H NMR (CDCl₃, 400MHz): 10.9 (br s, 1H), 6.86 (s, 1H), 6.64 (d, J=1.4 Hz, 1H), 4.77 (d,J=12.2 Hz, 1H), 4.65 (d, J=12.7 Hz,1H), 3.30−2.90 (m, 4H), 2.30−1.40 (m,7H).

Example 12

[0145]

(3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0146] A. 3-Bromo-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester.4-Bromo-thiophene-2-carbaldehyde (3.8 g, 20 mmol) was annulatedaccording to Procedure A to provide the title compound (1.2 g, 22%) as awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.48. ¹H NMR (CDCl₃,400 MHz): 9.58 (br s, 1H), 7.20 (s, 1H), 7.15 (d, J=1.5 Hz, 1H), 4.41(q, J=7.2 Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

[0147] B.(3-Bromo4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.3-Bromo4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (67 mg,0.24 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (65 mg,82% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₂H₁₄BrN₃OS,327.00; m/z found, 328.0 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 9.95 (br s,1H), 7.11 (s, 1H), 6.73 (d, J=1.8 Hz, 1H), 3.91 (t, J=5.1 Hz, 4H), 2.49(t, J=5.1 Hz, 4H), 2.34 (s, 3H).

Example 13

[0148]

(4-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone

[0149] A. 4-methyl-thiophene-2-carbaldehyde. A solution of3-methylthiophene (6.76 mL, 70 mmol) in ether (70 mL) was treated withn-butyllithium (2.5 M in hexanes, 28.6 mL, 71.4 mmol) at such a ratethat a slight reflux was maintained. The reaction mixture was heated toreflux for 15 min and then DMF (7.0 mL, 91 mmol) in ether (30 mL) wasadded. After stirring for 4 h, the reaction was quenched with additionof satd aq NH₄Cl (200 mL). The organic layer was separated, washed withbrine and then H₂O, dried over Na₂SO₄, and concentrated. Columnchromatography (SiO₂, 5-10% EtOAc/hexanes) provided a mixture of4-methyl-thiophene-2-carbaldehyde and 3-methyl-thiophene-2-carbaldehyde(4.4:1, 8.1 g, 92%) as a light yellow oil. TLC (silica, 10%EtOAc/hexanes): R_(f)=0.55. For 4-methyl-thiophene-2-carbaldehyde: ¹HNMR (CDCl₃, 400 MHz): 9.95 (s, 1H), 7.58 (d, J=1.2 Hz, 1H), 7.37−7.35(m, 1H), 2.32 (s, 3H). For 3-methyl-thiophene-2-carbaldehyde: ¹H NMR(CDCl₃, 400 MHz): 10.02 (s, 1H), 7.64 (d, J=4.6 Hz, 1H), 6.97 (d, J=4.6Hz, 1H), 2.58 (s, 3H).

[0150] B. 3-Methyl4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester.The mixture of 4-methyl-thiophene-2-carbaldehyde and3-methyl-thiophene-2-carbaldehyde (2.84 g, 22.5 mmol) was annulatedaccording to Procedure A to provide the title compound (2.5 g, 65%) as awhite solid. TLC (silica, 20% EtOAc/hexanes): R_(f)=0.45. ¹H NMR (CDCl₃,400 MHz): 9.95 (br s, 1H), 7.12 (d, J=1.9 Hz, 1H), 6.90 (d, J=1.2 Hz,1H), 4.39 (q, J=7.2 Hz, 2H), 2.35 (s, 3H), 1.39 (t, J=7.2 Hz, 3H).

[0151] C.(4-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone.3-Methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg,0.96 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (197 mg,78% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.4. MS (electrospray): exact mass calculated for C₁₃H₁₇N₃OS,263.11; m/z found, 264.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 11.10 (br s,1H), 6.76 (d, J=1.2 Hz, 1H), 6.69 (d, J=2.0 Hz, 1H), 3.94−3.90 (m, 4H),2.47 (t, J=5.1 Hz, 4H), 2.33 (s, 3H), 2.25 (s, 3H).

Example 14

[0152]

(2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0153] A. 2-Methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester.5-Methyl-furan-2-carbaldehyde (2.2 g, 20 mmol) was annulated accordingto Procedure A to provide the title compound (2.89 g, 75%) as a whitesolid. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.4. ¹H NMR (CDCl₃, 400MHz): 9.50 (br s, 1 H), 6.73 (s, 1H), 6.04 (s, 1H), 4.35 (q, J=7.2 Hz,2H), 2.37 (s, 3H), 1.35 (t, J=7.2 Hz, 3H).

[0154] B.(2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2-Methyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg,1.04 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (208 mg,81% for two steps) as a white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.35. MS (electrospray): exact mass calculated for C₁₃H₁₇N₃O₂,247.13; m/z found, 248.2 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 9.85 (br s,1H), 6.36 (s, 1H), 6.07 (s, 1H), 3.87 (t, J=5.0 Hz, 4H), 2.47 (t, J=5.2Hz, 4H), 2.39 (s, 3H), 2.33 (s, 3H).

Example 15

[0155]

(2,3-Dimethyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0156] A. 2,3-Dimethyl-4H-furo[3,2-b]pyrrole-5-carboxylic acid ethylester. 4,5-Dimethyl-furan-2-carbaldehyde (2.2 g, 18 mmol) was annulatedaccording to Procedure A to provide the title compound (1.76 g, 48%) asan off-white solid. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.35. ¹H NMR(CDCl₃, 400 MHz): 8.95 (br s, 1H), 6.69 (d, J=1.7 Hz, 1H), 4.32 (q,J=7.2 Hz, 2H), 2.33 (s, 3H), 2.08 (s, 3H), 1.37 (t, J=7.2 Hz, 3H).

[0157] B.(2,3-Dimethyl-4H-furo[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2,3-Dimethyl4H-furo[3,2-b]pyrrole-5-carboxylic acid ethyl ester (200 mg,0.97 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (190 mg,75% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.35. MS (electrospray): exact mass calculated for C₁₄H₁₉N₃O₂,261.15; m/z found, 261.8 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 9.95 (brs,1H), 6.32 (d, J=1.8 Hz, 1H), 3.88 (t, J=5.0 Hz, 4H), 2.47 (t, J=5.1Hz, 4H), 2.33 (s, 3H), 2.31 (s, 3H), 2.06 (s, 3H).

Example 16

[0158]

(2,3-Dimethyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone

[0159] A. 2,3-Dimethyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethylester. 4,5-Dimethyl-thiophene-2-carbaldehyde (2.0 g, 14 mmol) wasannulated according to Procedure A to provide the title compound (160mg, 5%) as a white solid. TLC (silica, 10% EtOAc/hexanes): R_(f)=0.40.¹H NMR (CDCl₃, 400 MHz): 9.50 br s, 1H), 7.05 (d, J=1.9 Hz, 1H), 4.36(q, J=7.2 Hz, 2H), 2.41 (s, 3H), 2.22 (s, 3H), 1.38 (t, J=7.2 Hz, 3H).

[0160] B.(2,3-Dimethyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.2,3-Dimethyl-4H-thieno[3,2-b]pyrrole-5-carboxylic acid ethyl ester (68mg, 0.30 mmol) was hydrolyzed (Procedure B) and then coupled withN-methylpiperazine (Procedure D) to provide the title compound (67 mg,80% for two steps) as an off-white solid. TLC (silica, 10% MeOH/CH₂Cl₂):R_(f)=0.35. MS (electrospray): exact mass calculated for C₁₄H₁₉N₃OS,277.12; m/z found, 278.1 [M+H]⁺. ¹H NMR (CDCl₃, 400 MHz): 10.95 (br s,1H), 6.63 (d, J=1.9 Hz, 1H), 3.92 (t, J=4.5 Hz, 4H), 2.46 (t, J=5.0 Hz,4H), 2.36 (s, 3H), 2.33 (s, 3H), 2.13 (s, 3H).

Examples 17-25

[0161] The following compounds were made according to the syntheticmethods outlined in Schemes 1-4:

EX Compound

[0162] 17(2,3-Dichloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0163] 18 (2-Methyl-4H-furo[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;

[0164] 19(3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;

[0165] 20(3-Bromo-4H-thieno[3,2-b]pyrrol-5-yl)-(3-methyl-piperazin-1-yl)-methanone;

[0166] 21(3-Methyl-4H-thieno[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;

[0167] 22(3-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-methanone;

[0168] 23(2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone;

[0169] 24(2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-piperazin-1-yl-methanone;and

[0170] 25(2,3-Dichloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-piperazin-1-yl)-methanone.

Biological Examples

[0171] Binding Assay on Recombinant Human Histamine H₄ Receptor

[0172] SK—N-MC cells or COS7 cells were transiently transfected withpH4R and grown in 150 cm² tissue culture dishes. Cells were washed withsaline solution, scraped with a cell scraper and collected bycentrifugation (1000 rpm, 5 min). Cell membranes were prepared byhomogenization of the cell pellet in 20 mM Tris-HCl with a polytrontissue homogenizer for 10 s at high speed. Homogenate was centrifuged at1000 rpm for 5 min at 4° C. The supernatant was then collected andcentrifuged at 20,000×g for 25 min at 4° C. The final pellet wasresuspended in 50 mM Tris-HCl. Cell membranes were incubated with³H-histamine (5-70 nM) in the presence or absence of excess histamine(10000 nM). Incubation occurred at room temperature for 45 min.Membranes were harvested by rapid filtration over Whatman GF/C filtersand washed 4 times with ice-cold 50 mM Tris HCl. Filters were thendried, mixed with scintillant and counted for radioactivity. SK—N-MC orCOS7 cells expressing human histamine H₄ receptor were used to measurethe affinity of binding of other compounds and their ability to displace³H-ligand binding by incubating the above-described reaction in thepresence of various concentrations of inhibitor or compound to betested. For competition binding studies using ³H-histamine, K_(i) valueswere calculated, based on an experimentally determined K_(D) value of 5nM and a ligand concentration of 5 nM, according to Y. -C. Cheng and W.H. Prusoff (Biochem. Pharmacol. 1973, 22(23):3099-3108):K_(i)=(IC₅₀)/(1+([L]/(K_(D))). BINDING ASSAY RESULTS EX K_(i) (nM) EXK_(i) (nM) 1 85 13 21 2 461 14 343 3 25 15 140 4 176 16 5 5 56 17 10 6840 18 770 7 125 19 410 8 343 20 980 9 733 21 80 10 40 22 161 11 715 233 12 56 24 30 25 5.5

[0173] Mast Cell Chemotaxis Assay

[0174] Mast cell accumulation in mucosal epithelia is a well-knowncharacteristic of allergic rhinitis and asthma. Transwells (Costar,Cambridge, Mass.) of a pore size 8 μm were coated with 100 μL of 100 μLof 100 ng/mL human fibronectin (Sigma) for 2 h at room temperature.After removal of the fibronectin, 600 μL of RPMI with 5% BSA, in thepresence of 10 μM histamine, was added to the bottom chamber. To testthe various histamine receptor (HR) antagonists, 10 μM and/or 1 μMsolutions of the test compounds were added to the top and bottomchambers. Mast cells (2×10⁵/well were added to the top chamber. Theplates were incubated for 37° C. Transwells were removed and the cellsin the bottom chamber were counted for sixty seconds using a flowcytometer. 10 μM HR Antagonist (μM): Binding Histamine 10 1 Assay EX %Inh Stdev % Inh Stdev K_(i) (nM) 3 106 4 103 0 25 4 <5 — <5 — 176 10 923 40 13 60 20 21 20 <5 — <5 — 980

[0175] Cell-Type Distribution of H₄ Expression

[0176] RNA was prepared from the different cells using an RNeasy kit(Qiagen, Valencia, Calif.) according to the manufacturer's instructions.RNA samples (5 μg) were run on an RNA gel and then transferred overnightto a nylon blot (Hybond, Amersham Pharmacia Biotech, Piscataway, N.J.).The blot was pre-hybridized with ExpressHyb solution (CLONTECH) for 30min at 68° C. The H₄ receptor DNA was labeled using the Rediprime II kit(Amersham Pharmacia Biotech). The blot was hybridized for 2 h at 68° C.,followed by one wash step (23 SSC and 0.05% SDS) of 40 min at roomtemperature, and a second wash step (0.13 SSC and 0.1% SDS) of 40 min at50° C. The blot was exposed to X-ray film at −70° C. with twointensifying screens overnight.

[0177] Results

[0178] The Northern Blot results indicate that the H₄ receptor isexpressed on bone marrow-derived mast cells (BMMC), peritoneal mastcells, and eosinophils. These positive results are consistent with thepublished literature (e.g. Oda et al., Nguyen et al., and Morse et al.in the Background section). However, the negative results of theNorthern Blot experiment, such as the finding of apparently nomeasurable levels of H₄ receptor expressed by neutrophils, differsomewhat from the above literature findings. This may be explained bythe different methodologies used. Accumulation of mast cells andeosinophils in affected tissues is one of the principal characteristicsof allergic rhinitis and asthma. Since H₄ receptor expression is limitedto these cell types; H₄ receptor signalling is likely to mediate theinfiltration of mast cells and eosinophils in response to histamine.Additional investigation may also clarify these issues. The followingtable reports the Cell-type Distribution of H₄ Expression by NorthernBlot. Species Cell Type H₄ Human Eosinophils + Immature Dendritic Cells− Mature Dendritic Cells − CD14⁺ Monocytes − CD4⁺ T Cells − CD8⁺ T Cells− B Cells − Neutrophils − Mouse/(Rat) Eosinophils + Peritoneal MastCells (Rat) + BMMC + BM Derived Macrophages − Peritoneal Macrophages −CD4⁺ T Cells − B Cells −

[0179] The Inhibition of Eosinophil Shape Change by Histamine H₄Receptor Antagonists

[0180] Eosinophil accumulation in sites of allergic reaction is awell-known characteristic of allergic rhinitis and asthma. This exampledemonstrates that histamine H₄ receptor antagonists can block the shapechange response in human eosinophils in response to histamine. Shapechange is a cellular characteristic that precedes eosinophil chemotaxis.

[0181] Methods

[0182] Human granulocytes were isolated from human blood by a Ficollgradient. The red blood cells were lysed with 5-10× Qiagen lysis bufferat room temperature for 5-7 min. Granulocytes were harvested and washedonce with FACS buffer. The cells were resuspended at a density of 2×10⁶cells/mL in reaction buffer. To test inhibition by specific histaminereceptor antagonists, 90 μL of the cell suspension (˜2×10⁵ cells) wasincubated with 10 μM of one of the various test compound solutions.After 30 min, 11 μL of one of the various concentrations of histaminewas added. Ten minutes later the cells were transferred to ice and fixedwith 250 μL of ice-cold fixative buffer (2% formaldehyde) for 1 min. Theshape change was quantitated using a gated autofluoescence forwardscatter assay (GAFS) (Byran et al., Am. J. Crit. Care Med. 2002,165:1602-1609).

[0183] Results—Histamine Mediates Eosinophil Shape Change Through H₄Receptor

[0184] The change in shape of eosinophils is due to cytoskeletal changesthat preceed chemotaxis and thus is a measure of chemotaxis. The data inthe following table show that histamine induces a dose-dependent shapechange in eosinophils. Histamine receptor (HR) antagonists were used tosort out which histamine receptor is responsible for the shape change.Antagonists specific for the histamine H₁ receptor (diphenhydramine) orthe H₂ receptor (ranatidine) did not alter the histamine-induced shapechange. However, a dual H₃/H₄ antagonist (thioperamide) and a specifichistamine H₄ receptor antagonist((5-Chloro-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone, K_(i)=5nM) inhibited histamine-induced eosinophil shape change with an IC₅₀ of1.5 and 0.27 μM, respectively. Histamine Fold Change (μM): 10 1 0.1 0.010 No HR 1.34 1.31 1.21 1.01 1.00 Antagonist 10 μM H₄ 1.09 1.05 1.05 1.011.00 Antagonist 10 μM 1.08 1.05 1.01 1.04 1.00 Thiop 10 μM 1.63 1.501.18 1.03 1.00 Diphen 10 μM 1.64 1.49 1.21 1.04 1.00 Ranat

[0185] The Inhibition of Eosinophil Chemotaxis by Histamine H₄ ReceptorAntagonists

[0186] Eosinophil accumulation in sites of allergic reaction is awell-known characteristic of allergic rhinitis and asthma. Eosinophilsare purified from human blood with standard methods. Chemotaxis assaysare carried out using transwells (Costar, Cambridge, Mass.) of a poresize 5 μm coated with 100 μL of 100 ng/mL human fibronectin (Sigma) for2 h at room temperature. After removal of the fibronectin, 600 μL ofRPMI with 5% BSA in the presence of histamine (ranging from 1.25-20 μM)is added to the bottom chamber. To test the various histamine receptorantagonists 10 μM of the test compounds can be added to the top andbottom chambers. Eosinophils will be added to the top chamber whereashistamine or chemotactic factors will be placed in the lower chamber.The plates are incubated for 3 h at 37° C. Transwells are removed andthe number of cells in the bottom chamber can be counted for 60 s usinga flow cytometer, or can be quantitated by using Giemsa staining.

[0187] The Inhibition of Zymosan-Induced Peritonitis in Mice byHistamine H₄ Receptor Antagonists

[0188] It has been demonstrated that histamine H₄ receptor antagonistscan block the peritonitis induced by zymosan, which is the insolublepolysaccharide component on the cell wall of Saccharomyces cerevisiae.This is commonly used to induce peritonitis in mice and appears to actin a mast cell-dependent manner. Compounds of the present invention canbe tested in such a model to demonstrate their use as anti-inflammatoryagents. At time 0 mice are given compound or PBS, either s.c. or p.o.Fifteen minutes later each mouse receives 1 mg zymosan A (Sigma) i.p.The mice are sacrificed 4 h later, and the peritoneal cavities arewashed with 3 mL of PBS containing 3 mM EDTA. The number of migratedleukocytes is determined by taking an aliquot (100 μL) of the lavagefluid and diluting 1:10 in Turk's solution (0.01% crystal violet in 3%acetic acid). The samples are then vortexed, and 10 μL of the stainedcell solution is placed in a Neubauer haemocytometer. Differential cellcounts are performed using a light microscope (Olympus B061). In view oftheir chromatic characteristics and their nucleus and cytoplasmappearance, polymorphonuclear leukocytes (PMN; >95% neutrophils) can beeasily identified. Treatment with zymosan increases the number ofneutrophils, which is representative of an inflammatory response.Treatment with H₄ receptor antagonist will block this incease.

[0189] Inhibition of Mast Cell Chemotaxis by H₄ Receptor Antagonist inan Animal Model of Asthma and Allergic Rhinitis

[0190] An animal model will be used to test the observation that mastcells accumulate in response to allergic inflammation and that this canbe blocked by H₄ receptor antagonists. Compounds of the presentinvention can be tested in this model to demonstrate their use astreatments for allergic rhinitis or asthma. Mice will be sensitized byintraperitoneal injection of ovalbumin/Alum (10 μg in 0.2 ml Al(OH)₃;2%) on Day 0 and Day 14. On Day 21 through 23 mice will be challenged byPBS or ovalbumin, and sacrificed 24 h after the last challenge on Day24. A section of the trachea will be removed and fixed in formalin.Paraffin embedding and longitudinal sectioning of tracheas will beperformed followed by staining of mast cells with toluidine blue.Alternatively, trachea will be frozen in OCT for frozen sectioning, andmast cells will be identified by IgE staining. Mast cells will bequantified as sub-mucosal or sub-epithelial depending on their locationwithin each tracheal section. Exposure to allergen should increase thenumber of sub-epithelial mast cells, and this effect will be blocked byH₄ receptor antagonists.

[0191] The features and advantages of the invention are apparent to oneof ordinary skill in the art. Based on this disclosure, including thesummary, detailed description, background, examples, and claims, one ofordinary skill in the art will be able to make modifications andadaptations to various conditions and usages. Publications describedherein are incorporated by reference in their entirety. These otherembodiments are also within the scope of the invention.

What is claimed is:
 1. A compound of formula (I):

Y is O or S; Z is O or S; n is 1 or 2; m is 1 or 2; n+m is 2 or 3; R¹ isH or C₁₋₆alkyl; R² is H, F, Cl, Br or C₁₋₆alkyl; R³ and R⁴ are,independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄alkyl(C₃₋₆cycloalkyl),cyano, —CF₃, —(CO)NR^(p)R^(q), —(CO)OR^(r), —CH₂NR^(p)R^(q) or—CH₂OR^(r); where R^(p), R^(q) and R^(r) are independently selected fromH, C₁₋₄alkyl, C₃₋₆cycloalkyl, phenyl, —C₁₋₂alkyl(C₃₋₆cycloalkyl), benzylor phenethyl, or R^(p) and R^(q) taken together with the nitrogen towhich they are attached, form a 4-7 membered heterocyclic ring with 0 or1 additional heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and whereany phenyl or alkyl or cycloalkyl moiety of the foregoing is optionallyand independently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; R⁵ and R⁶ are,independently, H or C₁₋₆alkyl; R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—O—R^(a)or —R^(e)—N(R^(c))(R^(d)), where R^(a) is H, cyano,—(C═O)N(R^(c))(R^(d)), —C(═NH)(NH₂), C₁₋₁₀alkyl, C₂₋₈alkenyl,C₃₋₈cycloalkyl, C₄₋₇heterocyclic radical or phenyl, where theC₄₋₇heterocyclic radical is attached at a carbon atom and contains oneof O, S, NH or NC₁₋₄alkyl, and optionally an additional NH or NC₁₋₆alkylin rings of 5 or 6 or 7 members, where R^(b) is C₁₋₈alkylene orC₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene or C₂₋₈alkenylene, whereR^(c) and R^(d) are each independently H, C₁₋₄alkyl, C₂₋₄alkenyl,C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkyl moiety of theforegoing is optionally and independently substituted with between 1 and3 substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, andC₁₋₃alkoxy; alternatively, R⁷ may be taken together with an adjacent R⁴as well as their carbon and nitrogen of attachment to form a 5, 6 or 7membered heterocyclic ring, with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and optionally and independentlysubstituted with between 1 and 3 substituents selected from C₁₋₃alkyl,halo, hydroxy, amino, and C₁₋₃alkoxy; R⁸ and R⁹ are, independently, H,F, Cl, Br, I, C₁₋₄alkyl, C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl,—OCH₂Ph, —CF₃, —OCF₃, —SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl,—OH, phenyl, benzyl, phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k)(where R^(t) is H or C₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl,—(S═(O)_(p))—C₁₋₄alkyl (wherein p is 0, 1 or 2), nitro, —SO₂NR^(l)R^(m)(wherein R^(l) and R^(m) are independently selected from H, C₁₋₄alkyl,phenyl, benzyl or phenethyl, or R^(l) and R^(m) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₄alkyl), —(C═O)NR^(l)R^(m), cyano or phenyl, where any phenyl oralkyl or cycloalkyl moiety of the foregoing is optionally andindependently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; and enantiomers,diastereomers and pharmaceutically acceptable salts and esters thereof,with the following provisos, that R⁶adjacent to N must be H where R⁴adjacent to N is other than H, that R⁷ is not —CH₂CH₂OH; and that wherethe core molecule is a 4H-furo, then one of R⁴ and R⁶ adjacent to N mustnot be methyl when the other is hydrogen unless R⁶ and R⁴ are takentogether to form a bridging moiety.
 2. A pharmaceutical compositioncontaining a compound of formula (I):

Y is O or S; Z is O or S; n is 1 or 2; m is 1 or 2; n+m is 2 or 3; R¹ isH or C₁₋₆alkyl; R² is H, F, Cl, Br or C₁₋₆alkyl; R³ and R⁴ are,independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄alkyl(C₃₋₆cycloalkyl),cyano, —CF₃, —(CO)NR^(p)R^(q), —(CO)OR^(r), —CH₂NR^(p)R^(q) or—CH₂OR^(r); where R^(p), R^(q) and R^(r) are independently selected fromH, C₁₋₄alkyl, C₃₋₆cycloalkyl, phenyl, —C₁₋₂alkyl(C₃₋₆cycloalkyl), benzylor phenethyl, or R^(p) and R^(q) taken together with the nitrogen towhich they are attached, form a 4-7 membered heterocyclic ring with 0 or1 additional heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and whereany phenyl or alkyl or cycloalkyl moiety of the foregoing is optionallyand independently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; R⁵ and R⁶ are,independently, H or C₁₋₆alkyl; R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—O—R^(a)or —R^(e)—N(R^(c))(R^(d)), where R^(a) is H, cyano,—(C═O)N(R^(c))(R^(d)), —C(═NH)(NH₂), C₁₋₁₀alkyl, C₂₋₈alkenyl,C₃₋₈cycloalkyl, C₄₋₇heterocyclic radical or phenyl, where theC₄₋₇heterocyclic radical is attached at a carbon atom and contains oneof O, S, NH or NC₁₋₄alkyl, and optionally an additional NH or NC₁₋₆alkylin rings of 5 or 6 or 7 members, where R^(b) is C₁₋₈alkylene orC₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene or C₂₋₈alkenylene, whereR^(c) and R^(d) are each independently H, C₁₋₄alkyl, C₂₋₄alkenyl,C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkyl moiety of theforegoing is optionally and independently substituted with between 1 and3 substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, andC₁₋₃alkoxy; alternatively, R⁷ may be taken together with an adjacent R⁴as well as their carbon and nitrogen of attachment to form a 5, 6 or 7membered heterocyclic ring, with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and optionally and independentlysubstituted with between 1 and 3 substituents selected from C₁₋₃alkyl,halo, hydroxy, amino, and C₁₋₃alkoxy; R⁸ and R⁹ are, independently, H,F, Cl, Br, I, C₁₋₄alkyl, C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl,—OCH₂Ph, —CF₃, —OCF₃, —SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl,—OH, phenyl, benzyl, phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k)(where R^(t) is H or C₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl,—(S═(O)_(p))—C₁₋₄alkyl (wherein p is 0, 1 or 2), nitro, —SO₂NR^(l)R^(m)(wherein R^(l) and R^(m) are independently selected from H, C₁₋₄alkyl,phenyl, benzyl or phenethyl, or R^(l) and R^(m) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₄alkyl), —(C═O)NR^(l)R^(m), cyano or phenyl, where any phenyl oralkyl or cycloalkyl moiety of the foregoing is optionally andindependently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; and enantiomers,diastereomers and pharmaceutically acceptable salts and esters thereof,with the following provisos, that R⁶ adjacent to N must be H where R⁴adjacent to N is other than H, that R⁷ is not —CH₂CH₂OH; and that wherethe core molecule is a 4H-furo, then one of R⁴ and R⁶ adjacent to N mustnot be methyl when the other is hydrogen unless R⁶ and R⁴ are takentogether to form a bridging moiety.
 3. A method for the treatment orprevention of H₄-mediated diseases and conditions comprising the step ofadministering to a patient in need of such treatment or prevention apharmaceutical composition containing an effective amount of a compoundof formula (I):

Y is O or S; Z is O or S; n is 1 or 2; m is 1 or 2; n+m is 2 or 3; R¹ isH or C₁₋₆alkyl; R² is H, F, Cl, Br or C₁₋₆alkyl; R³ and R⁴ are,independently, H, C₁₋₄alkyl, C₃₋₆cycloalkyl, C₁₋₄alkyl(C₃₋₆cycloalkyl),cyano, —CF₃, —(CO)NR^(p)R^(q), —(CO)OR^(r), —CH₂NR^(p)R^(q) or—CH₂OR^(r); where R^(p), R^(q) and R^(r) are independently selected fromH, C₁₋₄alkyl, C₃₋₆cycloalkyl, phenyl, —C₁₋₂alkyl(C₃₋₆cycloalkyl), benzylor phenethyl, or R^(p) and R^(q) taken together with the nitrogen towhich they are attached, form a 4-7 membered heterocyclic ring with 0 or1 additional heteroatoms selected from O, S, NH or NC₁₋₆alkyl, and whereany phenyl or alkyl or cycloalkyl moiety of the foregoing is optionallyand independently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; R⁵ and R⁶ are,independently, H or C₁₋₆alkyl; R⁷ is —R^(a), —R^(b)R^(a), —R^(e)—O—R^(a)or —R^(e)—N(R^(c))(R^(d)), where R^(a) is H, cyano,—(C═O)N(R^(c))(R^(d)), —C(═NH)(NH₂), C₁₋₁₀alkyl, C₂₋₈alkenyl,C₃₋₈cycloalkyl, C₄₋₇heterocyclic radical or phenyl, where theC₄₋₇heterocyclic radical is attached at a carbon atom and contains oneof O, S, NH or NC₁₋₄alkyl, and optionally an additional NH or NC₁₋₆alkylin rings of 5 or 6 or 7 members, where R^(b) is C₁₋₈alkylene orC₂₋₈alkenylene, where R^(e) is C₂₋₈alkylene or C₂₋₈alkenylene, whereR^(c) and R^(d) are each independently H, C₁₋₄alkyl, C₂₋₄alkenyl,C₃₋₆cycloalkyl or phenyl, or R^(c) and R^(d) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₆alkyl, and where any phenyl or alkyl or cycloalkyl moiety of theforegoing is optionally and independently substituted with between 1 and3 substituents selected from C₁₋₃alkyl, halo, hydroxy, amino, andC₁₋₃alkoxy; alternatively, R⁷ may be taken together with an adjacent R⁴as well as their carbon and nitrogen of attachment to form a 5, 6 or 7membered heterocyclic ring, with 0 or 1 additional heteroatoms selectedfrom O, S, NH or NC₁₋₆alkyl, and optionally and independentlysubstituted with between 1 and 3 substituents selected from C₁₋₃alkyl,halo, hydroxy, amino, and C₁₋₃alkoxy; R⁸ and R⁹ are, independently, H,F, Cl, Br, I, C₁₋₄alkyl, C₁₋₄alkoxy, —C₃₋₆cycloalkyl, —OC₃₋₆cycloalkyl,—OCH₂Ph, —CF₃, —OCF₃, —SCF₃, —(C═O)R^(k) (wherein R^(k) is H, C₁₋₄alkyl,—OH, phenyl, benzyl, phenethyl or C₁₋₆alkoxy), —(N—R^(t))(C═O)R^(k)(where R^(t) is H or C₁₋₄alkyl), —(N—R^(t))SO₂C₁₋₄alkyl,—(S═(O)_(p))—C₁₋₄alkyl (wherein p is 0, 1 or 2), nitro, —SO₂NR^(l)R^(m)(wherein R^(l) and R^(m) are independently selected from H, C₁₋₄alkyl,phenyl, benzyl or phenethyl, or R^(l) and R^(m) taken together with thenitrogen to which they are attached, form a 4-7 membered heterocyclicring with 0 or 1 additional heteroatoms selected from O, S, NH orNC₁₋₄alkyl), —(C═O)NR^(l)R^(m), cyano or phenyl, where any phenyl oralkyl or cycloalkyl moiety of the foregoing is optionally andindependently substituted with between 1 and 3 substituents selectedfrom C₁₋₃alkyl, halo, hydroxy, amino, and C₁₋₃alkoxy; and enantiomers,diastereomers and pharmaceutically acceptable salts and esters thereof,with the following provisos, that R⁶ adjacent to N must be H where R⁴adjacent to N is other than H, that R⁷ is not —CH₂CH₂OH; and that wherethe core molecule is a 4H-furo, then one of R⁴ and R⁶ adjacent to N mustnot be methyl when the other is hydrogen unless R⁶ and R⁴ are takentogether to form a bridging moiety.